Chemical reactions encompass molecular interactions such as the formation and cleavage of covalent bonds and ionic bonds; the association or dissociation of two or more chemical compounds; and changes in primary, secondary, tertiary, or quaternary structure. Chemical reactions include nonenzymatic and enzymatic reactions. Whether or not enzymes are present, a chemical reaction is usually made of several mechanistic steps or molecular interactions, including conformational changes, transition state formation, electron or proton donation/acceptance, and electron rearrangement. Typically, a series of chemical reactions provides a useful chemical product.
For example, in molecular biology, nested sets of deletions are used to create site-directed mutants which are used to probe the function of DNA segments in both structural and regulatory gene sequences. A collection of nested deletions within a gene allows the fine mapping of regions such as enhancers, promoters, and termination sites which are necessary for regulatory functions; and regions having structural function, such as those defining domains within proteins. It is desirable to create deletions which vary only slightly from each other, for example, by 10-20 base pairs. Existing methods for generating nested sets of deletions digest double stranded DNA with nucleases including restriction endonucleases, Bal31, pancreatic DNase I (DNase I), and Exonuclease III (Exo III).
Restriction endonucleases are used to partially digest a DNA template which contains multiple sites for a given restriction endonuclease. This method requires prior knowledge of restriction sites within a DNA template. Because restriction sites are not randomly distributed throughout a DNA template, many DNA templates will not contain sufficient or properly spaced restriction enzyme sites to generate a useful set of deletion mutants. This is particularly problematic when the mutants are intended to delineate the boundary of regulatory domains.
Turning to another endonuclease, Bal 31 digests double stranded linear DNA from both the 5' and 3' termini. To create a set of unidirectional mutants, a double stranded DNA template (plasmid, phage, or replicative form of M13) is linearized with a restriction enzyme which cleaves at one end of the target sequence. The linearized DNA is incubated with Bal 31. Varying time and the amount of enzyme respectively control the extent of digestion and the rate of digestion. Most commercial preparations of Bal 31 contain two distinct forms of the enzyme, a fast and a slow form, the latter being a proteolytic fragment of the former. The extent of digestion depends on the proportion of the two forms. Each batch of Bal 31 is therefore assayed to determine suitable digestion conditions.
Bal 31 is a processive enzyme which simultaneously degrades both the target DNA and the flanking vector DNA. Bal 31 activity varies with the primary structure of the DNA template; A-T rich regions are degraded faster than G-C rich regions. Recovery of the truncated target fragments and subcloning into an appropriate vector is required. The processive properties result in the generation of heterogeneous deletions. Bal 31 requires purification because it is inhibited by the presence of RNA.
Turning to a third enzyme, pancreatic DNase I will cut double stranded DNA templates at about the same location on both strands, in the presence of transition metal ions such as Mn.sup.2+ or Co.sup.2+. Incubation of closed circular DNA with DNase I generates a set of linear molecules which are cut at locations randomly dispersed throughout the target DNA. A portion of the starting material is never converted to the linear form. After a restriction enzyme cleaves at one end of the target sequence, the sequences are repaired using DNA polymerase, and recircularized. The fraction of clones recovered using this technique can be quite small. DNase I can generate deletions in target DNA contained within, for example, plasmid, phage, or the replicative form of M13 vectors [G. F. Hong, J. Mol. Biol. 158:539 (1982) and Methods Enzymol. 155:93 (1987); S. Labeit et al., Methods Enzymol. 155:166 (1987)].
One method of generating nested sets of deletions is digestion of double stranded DNA with Exo III [L.-H. Guo and R. Wu, Methods Enzymol. 100:60 (1983); S. Henikoff, Gene 23:351 (1984) and Methods Enzymol. 155:156 (1987)]. Exo III degrades double stranded DNA molecules in a 3' to 5' direction from either a 5' overhang or a blunt end. Nested deletions are generated by digesting the double stranded DNA with two restriction enzymes whose cleavage sites lie between one end of the target and the binding site for the universal sequencing primer on the vector. The restriction enzyme cutting nearest to the target DNA must generate either a blunt-end or a 5' overhang. The other enzyme must generate a 3' overhang. As Exo III cannot degrade DNA having a 3' overhang, digestion of the doubly restricted molecule proceeds in a unidirectional manner. Following Exo III digestion for varying lengths of time, the single-stranded regions are removed with a single-strand nuclease such as Mung Bean nuclease. The DNA is then repaired and recircularized. The extent of Exo III digestion is controlled by varying the length of the incubation period. In addition, the temperature may be lowered to decrease the rate of digestion [G. Murphy, in DNA sequencing Protocols, H. G. Griffin and A. M. Griffin, eds., Humana Press (1993) p. 58]. This method requires two restriction enzymes which satisfy the above conditions and which do not cut within the target DNA. This requirement is difficult to satisfy when the target DNA is long.